One of the major unsolved mysteries in tumor biology is the mechanism by which tumor cells in vivo exit from the cell cycle in a reversible fashion. An implicit assumption through decades of research is that limitations of nutrient supply, either by limited penetration into the cell mass or restricted blood flow to local regions, create a stressful microenvironment which induces cells to arrest their cell cycle transit. Due to well-known limitations of experimental tumors for such mechanistic studies, we are using the multicellular tumor spheroid model for the majority of this project. Spheroids are ideally suited for such studies, both because of their symmetrical arrangement of microenvironmental and cellular proliferation gradients, and because of our unique ability to experimentally exploit this symmetry. Specifically, we can isolate intact, viable cells from known locations within the spheroid microenvironment for detailed study of the molecular changes ass ociated with cell cycle arrest. In order to provide a link between this in vitro system and the in vivo situation we will determine whether our proposed mechanism is operative in actual tumors. We are pursuing four Specific Aims: 1) to determine the molecular basis for cell cycle arrest in multicellular spheroids; 2) to determine if the same molecular mechanisms are operative in tumors in vivo; 3) to identify the microenvironmental signal(s) which induce cell cycle arrest in spheroids; and 4) to determine the interaction between radiation- and microenvironmentally-induced cell cycle arrest. Flow analysis is used both for routine DNA content analysis, and also for determining the uptake of bromodeoxyuridine by means of a dual-label DNA analysis technique. These cell cycle data are critical for comparison with our molecular analysis. We are also pursuing the measurement of cyclin and cyclin-dependent kinase expression by flow using fluorescently-tagged antibodies.